Borrelia burgdorferi causes Lyme disease, the most common vector-borne disease in the United States. The spirochete has to change its gene expression during its life cycle in order to survive in the different environments that it encounters. p35 and p37 are unrelated genes with a coincident pattern of antibody response, peaking at around day 30 of infection and declining afterwards. Both genes are expressed early during infection, and may play a role in dissemination and colonization of the joints upon entering the mammalian host and cause arthritis. This project propose the use of genetic manipulation of clonal infectious B. burgdorferi in order to gain insight into the function of p35 and p37. Both genes will be inactivated by homologous recombination with an interrupted copy of the gene by insertion of a mutated gyrB gene (gyrBr), which confers resistance to coumermycin A1, the only selectable marker used successfully in non-infectious B. burgdorferi. The resultant mutants will be used to infect mice and study their ability to disseminate in the murine host, as well as their capacity to complete a natural cycle and cause disease. The ability to genetically manipulate infectious B. burgdorferi opens the possibility to study spirochetal gene function in natural conditions, and marks a forward step from gene description to the study of gene function in vivo.